The proposed work focuses on the development of advanced methods in nuclear magnetic resonance (NMR) spectroscopy for metabolomics-based studies. The rapidly expanding field of metabolomics provides new information on biological perturbations based on multiple changes in small molecule metabolite signals. New advances in NMR spectroscopy and their combination with mass spectrometry (MS) and multivariate statistical methods provide a promising approach for a number of biological applications including early disease diagnosis, treatment monitoring, drug development and basic investigations in systems biology. While the metabolome is known to provide an instantaneous snap-shot of health status, the identification and validation of potential biomarkers of health and disease is challenging due to the complexity of their overlapping signals in biological samples. Over the past several years, we have developed a new set of advanced NMR tools that can be brought to bear on this problem, with the promise that we can significantly increase the ability of NMR to dissect the complex samples and identify sensitive and specific metabolite biomarker candidates. In particular we propose three advanced NMR methods that are expected to improve the limit of detection of metabolites in biofluids by 10-fold and increase the number of NMR-detectible metabolites by a similar factor. This development would change the current paradigm in NMR-based metabolomics and provide an even stronger complement to current MS-based methods. Validation of these methods on a set of commercial serum samples and a comparison of these new approaches to current methods are also discussed. If this work is successful, we will have laid the foundation for much improved metabolomics studies for applications ranging from early disease detection and therapy monitoring to basic studies of systems biology. PUBLIC HEALTH RELEVANCE This project seeks to develop new and advanced methods for identifying biomarkers of disease in serum and urine samples. When fully developed these approaches could be used to detect small molecules in the blood or urine that indicate the onset of various diseases.